Background Signal Intensity Research Articles

200 EFFECTS OF MELATONIN ON REACTIVE OXYGEN SPECIES GENERATION AND ACQUISITION OF EMBRYONIC DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN VITRO

Reactive oxygen species (ROS) are produced under normal culture conditions, but when production increases, it generates a harmful condition called oxidative stress (OS), leading to apoptosis and developmental blocks. Addition of antioxidants as melatonin to culture media has been used to minimize the effects of OS. Our hypothesis was that melatonin could improve oocyte in vitro maturation (IVM) and protect oocytes from ROS under a standard culture condition, thus increasing embryonic development. To test, cumulus-oocyte complexes were matured in TCM-199 with bicarbonate, 0.5 mg mL–1 of FSH, 100 IU mL–1 of hCG, and 10% FCS without supplementation (control group) or supplemented with 10–5 (MT5), 10–7 (MT7) or 10–9 (MT9) M melatonin for 22 h at 38.5°C and 5% CO2 in air. After IVM, a sample of oocytes (control, n = 59; MT5, n = 64; MT7, n = 77; MT9, n = 57) was stained with 1 µg mL–1 Hoechst 33342 to assess the nuclear maturation, and oocytes were classified as being in the stages of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I or telophase I (A/T), or metaphase II (MII). To determine the intracellular ROS levels, other matured oocytes (control, n = 46; MT5, n = 59; MT7, n = 51; MT9, n = 61) were stained with 5 µM CellROX®Green (Molecular Probes, Eugene, OR, USA) and were evaluated immediately under an epifluorescence inverted microscope (excitation 485 nm; emission 520 nm). The images were recorded and further analysed by Q-Capture Pro image software. After subtraction of the background signal intensity from the measured fluorescent signal intensity values, 1 group was chosen as a calibrator (control group), and each treatment value was divided by the mean calibrator value to generate the relative expression level (in arbitrary fluorescence units). Finally, another sample of matured oocytes (control, n = 188; MT5, n = 173; MT7, n = 180; MT9, n = 178) was submitted to IVF (Day = 0), and the presumptive zygotes were cultured in SOF at 38.5°C and 5% CO2 in air, for up to 7 days. The cleavage rates and embryonic development were evaluated at Days 3 and 7 of IVC, respectively. Data were analysed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as mean ± SEM. There was no effect (P > 0.05) of different concentrations of MT on nuclear status of matured oocytes, as we found no differences in the rates of GV (0% to 5.3% ± 3.4), GVBD (5.4% ± 3.2 to 16.3% ± 5.0), MI (1.7% ± 1.7 to 3.2% ± 3.2), AI/TI (0% to 5.4% ± 3.4), and MII (74.8% ± 2.7 to 87.5% ± 3.7). The cleavage rates did not differ (P > 0.05) among treatments (76.7% ± 4.4 to 83.8% ± 2.7), as well as the embryonic development to the blastocyst stage (31.2% ± 1.9 to 43.7% ± 5.7). The intracellular ROS levels decreased significantly (P < 0.05) in the MT9 group (0.75 ± 0.03) in comparison to Control (1.0 ± 0.06), MT5 (0.97 ± 0.05) and MT7 (0.94 ± 0.05). In conclusion, supplementation with 10–9 M melatonin during IVM reduced the intracellular ROS levels of oocytes without interfering with the nuclear maturation and the subsequent embryonic development to the blastocyst stage. Financial support was provided by FAPESP (#2013/07382–6).

Apparent diffusion coefficient-dependent voxelwise computed diffusion-weighted imaging: An approach for improving SNR and reducingT2shine-through effects

To introduce and evaluate a method for signal-to-noise ratio (SNR) improvement and T2 shine-through effect reduction in diffusion-weighted magnetic resonance imaging (DWI). The proposed method uses quantitative information given by the voxel apparent diffusion coefficient (ADC) to derive voxelwise-computed DWI (vcDWI). Behavior of signal intensity variations was simulated and correlated with measurements using a dedicated phantom for DWI allowing for independent adjustment of T2 -relaxivity and diffusivity. Signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were measured and compared to the method of computed DWI (cDWI). Image signal was correlated with ADCs to appreciate the extent of T2 shine-through effects. Additionally, the proposed method was retrospectively applied to whole-body DWI data of 20 patients with metastatic malignancies. vcDWI was compared to cDWI and measured DWI with respect to image quality, lesion detectability, and lesion diffusivity assessment. Theoretically predicted signal intensity variations showed a high correlation with measured phantom data (r > 0.96). The proposed method yielded lower background signal intensity variation and higher contrast (+144%) and CNR (+358%) for diffusion-restricted phantom compartments than cDWI. Signal intensities of vcDWI showed an increased inverse correlation with phantom ADC values compared to cDWI (r = -0.86 vs. r = -0.73). Application to patient data showed higher image quality (P < 0.001) and lesion detectability (P = 0.011) using vcDWI compared to cDWI, and higher confidence for the correct identification of diffusion-restricted lesions compared to measured DWI (80/80 vs. 60/81; P = 0.013). vcDWI is a promising approach for the reduction of T2 shine-through effects and improvement of SNR and CNR in DWI. The clinical significance of these improvements, especially regarding lesion detection, needs to be evaluated in larger prospective clinical studies.

TWO FACTORS INFLUENCING DOSE RECONSTRUCTION IN LOW DOSE RANGE: THE VARIABILITY OF BKG INTENSITY ON ONE INDIVIDUAL AND WATER CONTENT.

A fast and accurate retrospective dosimetry method for the triage is very important in radiation accidents. Electron paramagnetic resonance (EPR) fingernail dosimetry is a promising way to estimate radiation dose. This article presents two factors influencing dose reconstruction in low dose range: the variability of background signal (BKG) intensity on one individual and water content. Comparing the EPR spectrum of dried and humidified fingernail samples, it is necessary to add a procedure of dehydration before EPR measurements, so as to eliminate the deviation caused by water content. Besides, the BKGs of different fingers' nails are not the same as researchers thought previously, and the difference between maximum and minimum BKG intensities of one individual can reach 55.89 %. Meanwhile, the variability of the BKG intensity among individuals is large enough to impact precise dose reconstruction. Water within fingernails and instability of BKG are two reasons that cause the inaccuracy of radiation dose reconstruction in low-dosage level.

Determination of U–Pb Ages for Young Zircons using Laser Ablation‐ICP‐Mass Spectrometry Coupled with an Ion Detection Attenuator Device

We have developed new analytical procedures to measure precise and accurate 238U–206Pb and 235U–207Pb ages for young (~ 1 Ma) zircons using laser ablation‐ICP‐mass spectrometry. For young zircons, both careful correction for the background counts and analysis of very small Pb/U ratios (i.e., 206Pb/238U &lt; 0.00016 and 207Pb/235U &lt; 0.0001 for 1 Ma zircons) are highly desired. For the correction of the background, the contribution of the background signal intensities for the analytes, especially for the residual signal intensities for 206Pb and 207Pb, was defined through laser ablation of synthesised zircons (ablation blank) containing negligible Pb. The measured signal intensities for 202Hg, 206Pb and 207Pb signals obtained by the ablation blank were slightly higher than those obtained by data acquisition without laser ablation (gas blank). For the wider dynamic range measurements on Pb/U ratios, an attenuator device for the ion detection system was employed to extend the capability to monitor high‐intensity signals (i.e., &gt; 3 Mcps). Through the attenuator device, the ion currents were reduced to 1/450 of the signal intensity without the attenuator. Because the switching time for the attenuator was shorter than 1 ms, signal intensities for only specific isotopes could be reduced. With attenuation of the 238U signal, counting statistics on 206Pb and 207Pb isotopes could be improved and counting loss on the 238U signal could be minimised. To demonstrate the reliability of this new analytical technique, 238U–206Pb and 235U–207Pb ages for three young zircon samples (collected from Osaka Group Pink Volcanic Ash, Kirigamine and Bishop Tuff) were measured. The data presented here demonstrate clearly that the present technique could become a major analytical tool for in situ U–Pb age determination of young zircons (~ 1 Ma).

Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids

Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if “thinner” TLC layers are used.

An extremely simple method for fabricating 3D protein microarrays with an anti-fouling background and high protein capacity.

Protein microarrays have become vital tools for various applications in biomedicine and bio-analysis during the past decade. The intense requirements for a lower detection limit and industrialization in this area have resulted in a persistent pursuit to fabricate protein microarrays with a low background and high signal intensity via simple methods. Here, we report on an extremely simple strategy to create three-dimensional (3D) protein microarrays with an anti-fouling background and a high protein capacity by photo-induced surface sequential controlled/living graft polymerization developed in our lab. According to this strategy, "dormant" groups of isopropyl thioxanthone semipinacol (ITXSP) were first introduced to a polymeric substrate through ultraviolet (UV)-induced surface abstraction of hydrogen, followed by a coupling reaction. Under visible light irradiation, the ITXSP groups were photolyzed to initiate surface living graft polymerization of poly(ethylene glycol) methyl methacrylate (PEGMMA), thus introducing PEG brushes to the substrate to generate a full anti-fouling background. Due to the living nature of this graft polymerization, there were still ITXSP groups on the chain ends of the PEG brushes. Therefore, by in situ secondary living graft cross-linking copolymerization of glycidyl methacrylate (GMA) and polyethylene glycol diacrylate (PEGDA), we could finally plant height-controllable cylinder microarrays of a 3D PEG network containing reactive epoxy groups onto the PEG brushes. Through a commonly used reaction of amine and epoxy groups, the proteins could readily be covalently immobilized onto the microarrays. This delicate design aims to overcome two universal limitations in protein microarrays: a full anti-fouling background can effectively eliminate noise caused by non-specific absorption and a 3D reactive network provides a larger protein-loading capacity to improve signal intensity. The results of non-specific protein absorption tests demonstrated that the introduction of PEG brushes greatly improved the anti-fouling properties of the pristine low-density polyethylene (LDPE), for which the absorption to bovine serum albumin was reduced by 83.3%. Moreover, the 3D protein microarrays exhibited a higher protein capacity than the controls to which were attached the same protein on PGMA brushes and monolayer epoxy functional groups. The 3D protein microarrays were used to test the immunoglobulin G (IgG) concentration in human serum, suggesting that they could be used for biomedical diagnosis, which indicates that more potential bio-applications could be developed for these protein microarrays in the future.

A non-invasive method for in vivo skin volatile compounds sampling

The use of volatile organic compounds (VOCs) emanating from human skin presents great potential for skin disease diagnosis. These compounds are emitted at very low concentrations. Thus, the sampling preparation step needs to be implemented before gas chromatography–mass spectrometry (GC–MS) analysis. In this work, a simple, non-invasive headspace sampling method for volatile compounds emanating from human skin is presented, using thin film as the extraction phase format.The proposed method was evaluated in terms of reproducibility, membrane size, extraction mode and storage conditions. First, the in vial sampling showed an intra- and inter-membrane RSD% less than 9.8% and 8.2%, respectively, which demonstrated that this home-made skin volatiles sampling device was highly reproducible with regard to intra-, inter-membrane sampling. The in vivo sampling was influenced not only by the skin metabolic status, but also by environmental conditions. The developed sampling set-up (or “membrane sandwich”) was used to compare two different modes of sampling: headspace and direct sampling. Results demonstrated that headspace sampling had significantly reduced background signal intensity, indicating minimized contamination from the skin surface. In addition, membrane storage conditions both before and after sampling were fully investigated. Membranes stored in dry ice for up to 72h after collection were tested and showed no or minimal change in volatile profiles.This novel skin volatile compounds sampling approach coupled with gas chromatography–mass spectrometry (GC–MS) can achieve reproducible analysis. This technique was applied to identify the biomarkers of garlic intake and alcohol ingestion. Dimethyl sulphone, allyl methyl sulfide and allyl mercaptan, as metabolites of garlic intake, were detected. In addition, alcohol released from skin was also detected using our “membrane-sandwich” sampling. Using the same approach, we analyzed skin VOCs from upper back, forearm and back thigh regions of the body. Our results show that different body locations share a number of common compounds (27/99). The area with most compounds detected was the upper back skin region, where the density of sebaceous glands is the highest.

Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pKa value.

Quantitative analysis of the breath-holding half-Fourier acquisition single-shot turbo spin-echo technique in abdominal MRI

A consecutive series of 50 patients (28 males and 22 females) who underwent hepatic magnetic resonance imaging (MRI) from August to December 2011 were enrolled in this study. The appropriate parameters for abdominal MRI scans were determined by comparing the images (TE = 90 and 128 msec) produced using the half-Fourier acquisition single-shot turbo spin-echo (HASTE) technique at different signal acquisition times. The patients consisted of 15 normal patients, 25 patients with a hepatoma and 10 patients with a hemangioma. The TE in a single patient was set to either 90 msec or 128 msec. This was followed by measurements using the four normal rendering methods of the biliary tract system and the background signal intensity using the maximal signal intensity techniques in the liver, spleen, pancreas, gallbladder, fat, muscles and hemangioma. The signal-to-noise and the contrast-to-noise ratios were obtained. The image quality was assessed subjectively, and the results were compared. The signal-to-noise and the contrast-to-noise ratios were significantly higher at TE = 128 msec than at TE = 90 when diseases of the liver, spleen, pancreas, gallbladder, and fat and muscles, hepatocellular carcinomas and hemangiomas, and rendering the hepatobiliary tract system based on the maximum signal intensity technique were involved (p < 0.05). In addition, the presence of artifacts, the image clarity and the overall image quality were excellent at TE = 128 msec (p < 0.05). In abdominal MRI, the breath-hold half-Fourier acquisition single-shot turbo spin-echo (HASTE) was found to be effective in illustrating the abdominal organs for TE = 128 msec. Overall, the image quality at TE = 128 msec was better than that at TE = 90 msec due to the improved signal-to-noise (SNR) and contrast-to-noise (CNR) ratios. Overall, the HASTE technique for abdominal MRI based on a high-magnetic field (3.0 T) at a TE of 128 msec can provide useful data.

Dispersion of hyperenhancement in late gadolinium enhancement cardiovascular magnetic resonance measured with Moran's I is associated with a decrement in LVEF 6 months after cardiotoxic chemotherapy

Background In animals and human subjects, an increase in background signal intensity observed on late gadolinium enhanced (LGE-SI) images is associated with a decrement in left ventricular ejection fraction (LVEF) during receipt of anthracycline chemotherapy. Moran’ sIs tatistic is a measurement of spatial dispersion of hyperenhanced voxels relative to the mean myocardial LGE-SI, ranging from highly clustered (I=+1) to highly diffuse (I=-1) (Figure 1). We hypothesize that a change in the distribution of hyperenhanced voxels (due to the development of high signal “micro clusters”) is associated with a decrement in LVEF after cardiotoxic chemotherapy. Methods We performed a prospective, extramurally-funded longitudinal cohort study of 51 participants (43 women, 8 men; aged 52±2 years) scheduled to receive 3 to 4 months of potentially cardiotoxic chemotherapy (anthracycline or trastuzumab) for treatment of breast cancer or hematologic malignancy. Before and then 3 and 6 months after chemotherapy initiation, participants underwent cardiovascular magnetic resonance (CMR) assessments of LVEF, LGE-SI, and Moran’s I statistic determined by personnel blinded to participant identifiers and all other aspects of the analyses. Results were analyzed using paired Student’s t-tests to test for a difference between baseline and subsequent examinations, and one-way ANOVA to test for trending change. All values are reported as mean ± standard deviation with p-values<0.05 considered statistically signficant.

Bone metastases from prostate, breast and multiple myeloma: differences in lesion conspicuity at short-tau inversion recovery and diffusion-weighted MRI

The objective of this study was to compare the relative conspicuity of bone metastases on short-tau inversion recovery (STIR) and diffusion-weighted MRI (DWI) whole-body MR sequences for breast, prostate and myeloma malignancies. 44 whole-body MRI scans were reviewed retrospectively (coronal T(1) weighted, STIR and DWI with b=800). On each scan, up to four of the largest bone lesions were identified on T(1) weighting, and the region of interest signal intensity was measured on STIR and DWI, as well as the background signal intensity. The mean lesion signal to background ratio was calculated for each patient and then for each malignancy group. In prostate cancer patients, the DWI signal/background ratio was greater than that of STIR in 22 out of 24 patients (mean DWI lesion/background ratio 3.91, mean STIR lesion/background ratio 2.31; p=0.0001). In multiple myeloma, the DWI ratio was higher in 6/7 patients (DWI group mean ratio 7.59, STIR group mean ratio 3.7; p=0.0366). In 13 breast cancer patients, mean STIR and DWI signal/background were similar (DWI group mean ratio 4.13, group mean STIR ratio 4.26; p=0.8587). Bone lesion conspicuity measured by lesion/background signal intensity was higher on DWI b=800 than on STIR in patients with prostate cancer and multiple myeloma. DWI should be used in whole-body MR oncology protocols in these conditions to maximise lesion detection.

Locoregional Chemoembolic Delivery: Prediction With Transcatheter Intraarterial Perfusion MRI

To our knowledge there is currently no quantitative preprocedural method for predicting the distribution and selectivity of delivery of chemoembolic material during trans-arterial chemoembolization. Transcatheter intraarterial perfusion MRI has been developed as a method of quantifying hepatic arterial perfusion. The purpose of this study was to investigate whether findings at transcatheter intraarterial perfusion MRI before chemoembolization can be used to predict uptake of the chemoembolic material delivered during chemoembolization. We compared quantitative prechemoembolization transcatheter intraarterial perfusion MRI parameters with analogous postchemoembolization CT chemoembolic distribution parameters and analyzed correlation using the Pearson correlation coefficient. These MRI and CT parameters included volume of distribution (a metric for volumetric liver perfusion or therapeutic agent delivery) and chemoembolic delivery selectivity factor (a ratio of volume-normalized tumor to background signal intensity that indicates the selectivity of chemoembolic delivery). Twenty-four hepatocellular carcinomas were targeted in 18 patients (14 men, four women; mean age, 66 years), and segmental or lobar chemoembolization with intraprocedural transcatheter intraarterial perfusion MRI was successful in all 18. Transcatheter intraarterial perfusion MRI and CT volume of distribution did not differ significantly (MRI, 233 cm(3); CT, 235 cm(3); p = 0.857). Transcatheter intraarterial perfusion MRI selectivity factor was an underestimate of CT selectivity factor (MRI, 0.20; CT, 0.25; p = 0.005). Prechemoembolization transcatheter intraarterial perfusion MRI and postchemoembolization CT volume of distribution (r = 0.93; p < 0.001) and selectivity factor (r = 0.95; p < 0.001) showed significant correlation. Tumor perfusion measured with transcatheter intraarterial perfusion MRI is predictive of uptake of chemoembolic material before delivery. This MRI technique may have utility as a method of quantifying delivery of the therapeutic agent during chemoembolization and, potentially, other liver-directed locoregional therapies.

T1-Weighted MRI shows stage-dependent substantia nigra signal loss in Parkinson's disease

Depigmentation of the substantia nigra is a conspicuous pathological feature of Parkinson's disease and related to a loss of neuromelanin. Similar to melanin, neuromelanin has paramagnetic properties resulting in signal increase on specific T1-weighted magnetic resonance imaging. The aim of this study was to assess signal changes in the substantia nigra in patients with Parkinson's disease using an optimized neuromelanin-sensitive T1 scan. Ten patients with Parkinson's disease and 12 matched controls underwent high-resolution T1-weighted magnetic resonance imaging with magnetization transfer effect at 3T. The size and signal intensity of the substantia nigra pars compacta were determined as the number of pixels with signal intensity higher than background signal intensity+3 standard deviations and regional contrast ratio. Patients were subclassified as early stage (n=6) and late stage (n=4) using the Unified Parkinson's Disease Rating Scale and the Hoehn and Yahr Parkinson's disease staging scale. The T1 hyperintense area in the substantia nigra was substantially smaller in patients compared with controls (-60%, P<.01), and contrast was reduced (-3%, P<.05). Size reduction was even more pronounced in more advanced disease (-78%) than in early-stage disease (-47%). We present preliminary findings using a modified T1-weighted magnetic resonance imaging technique showing stage-dependent substantia nigra signal reduction in Parkinson's disease as a putative marker of neuromelanin loss. Our data suggest that reduction in the size of neuromelanin-rich substantia nigra correlates well with postmortem observations of dopaminergic neuron loss. Further validation of our results could potentially lead to development of a new biomarker of disease progression in Parkinson's disease.

Gadopentetate dimeglumine-enhanced MR cholangiopancreatography in infants with cholestasis

Biliary atresia (BA) is a progressive, obliterative cholangiopathy that occurs in neonates with hepatic portoenterostomy the treatment of choice, but early surgery is important for optimum outcomes. MRI, including MR cholangiopancreatography (MRCP) may be a diagnostically useful alternative to US, but the heavily T2-weighted sequences used include not only bile duct signals, but also other heterogeneously high signal intensities from surrounding structures. To evaluate the effects of gadolinium when used to decrease background signal intensity on T2-weighted MR cholangiopancreatography (MRCP) in infants and to evaluate the qualitative improvement of the depiction of the common bile duct (CBD) for evaluating neonatal cholestasis. Our Institutional Review Board approved this prospective study. MRCP was performed with gadopentetate dimeglumine injection using a 1.5-T scanner. Pre- and postcontrast MRCP images were compared. Forty-nine infants (male:female = 21:28; age 0-12 months, mean 2.3) were included. The final diagnoses were biliary atresia (BA) in 28 cases and non-BA in 21. Quantitative analysis was conducted using region-of-interest measurements of mean signal intensities of the liver, pancreatic head and gallbladder (if defined). Qualitative analysis was performed by four radiologists who subjectively scored image confidence in the presence of CBD on a 4-point scale (0 for definitely absent, 1 for probably absent, 2 for probably present, and 3 for definitely present). The signal-to-noise ratios were significantly decreased in the liver and pancreatic head after contrast medium enhancement (mean 5.7→4.0 in liver and mean 44.9→12.7 in the pancreatic head; P < 0.0001), and this finding was constant in both the BA and the non-BA group. The mean confidence score in the presence of CBD decreased in the BA group (0.9→0.5; P < 0.0001), but did not change significantly in the non-BA group (2.0→2.1; P = 0.459) after contrast medium enhancement. Both intra- and interobserver agreement was higher after contrast medium enhancement (P = 0.046). Gadopentetate dimeglumine-enhanced MRCP increased the diagnostic confidence of absence of the CBD in cholestatic infants with increased intra- and interobserver agreement.

Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics

Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory. Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed. With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after double-staining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining. We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase ( Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

A scFv Antibody-Based Immunoaffinity Chromatography Column for Clean-Up of Bisphenol A-Contaminated Water Samples

This is a report on the development of immunoaffinity chromatography using a column of silica gel with an immobilized single-chain variable fragment (scFv) antibody specific to bisphenol A (BPA) for cleanup of BPA-contaminated water samples. The BBA-2187 scFv antibody specific to BPA was purified from the periplasmic fractions of the recombinant Escherichia coli. After a sample of BPA-contaminated river water was applied to the immunoaffinity column, the background signal intensity observed in high-performance liquid chromatography (HPLC) analysis of the eluates was markedly lower than that observed in HPLC analysis of the eluates from an Oasis HLB cartridge treated with the same sample. The immunoaffinity column efficiently concentrated BPA from actual river water samples with different matrices. Our results demonstrate that the immunoaffinity column with immobilized BBA-2187 scFv antibody is efficient for the cleanup of BPA-contaminated water samples from different sources.

Abstract 3418: Infarct-Related Myocardial Edema Detected by Diffusion-Weighted Imaging; Comparison with T2-Weighted imaging

Background: Cardiac MRI permits diagnosis with acute myocardial infarction (AMI) by detection of infarct-related myocardial edema (IRME) on black-blood T2-weighted images (T2WIs), and myocardial scar on delayed-enhanced images (DEs). Diffusion-weighted image (DWI) is an established method for detection of cerebral infarction edema, but few reports are available on detection of IRME. Objective: To detect IRME using DWIs, and compare the contrast-to-noise ratio (CNR) with T2WIs. Methods: Cardiac MRI was performed on a 1.5T scanner (SIEMENS Avanto, Erlangen, Germany) in 12 AMI patients within 14 days after successful reperfusion. T2WIs were acquired using a double inversion-recovery fast-spin echo sequence. DWIs were acquired using combined cardiac and respiratory gated single-shot echo-planar sequence with a b-value of 50 s/mm 2 . DEs were acquired using an inversion-recovery segmented gradient-echo sequence, fifteen minutes after administering 0.15 mmol/kg gadolinium-DTPA. CNR was calculated in the T2WIs and DWIs using the following formula: CNR = (signal intensity[ME]-signal intensity[M]) / SD(B), where ME = myocardial edema, M = myocardium, and SD(B) =standard deviation of background signal intensity. Results: IRME was detected on T2WIs, and myocardial scar on DEs in all patients. DWIs revealed an area of significantly increased signal intensity in the same region identified as IRME on T2WIs in all patients. The CNR of DWIs (78.9 ± 44.5) was significantly higher than those of T2WIs (31.5 ± 23.3) (p = 0.0001). Conclusion: Our findings indicate that low b-value DWI provides better contrast in infarct-related myocardial edema than T2WIs.

ToF-SIMS characterisation of diterpenoic acids after chromatographic separation

Microcolumn liquid chromatography (μHPLC) coupled on-line with time of flight secondary ion mass spectrometry (ToF-SIMS) was applied for mixture of diterpenoic acids (abietic, gibberellic and kaurenoic) analysis. Chromatographic effluent, with analytes separated, was carried out directly onto different, ToF-SIMS compatible surface substrates, for further ToF-SIMS analysis. Silica gel Si 60, aluminium backplate modified Si 60, monolithic silica gel and Raman spectroscopy chromatographic thin layers were used as the deposition substrates in this experiment. By ToF-SIMS surface imaging the deposition trace picture has been obtained. Effluent deposition surface area was scanned for diterpenoic acid fragment mass values based on mass spectrometric library. Measured ToF-SIMS dataset of fragment abundance and intensities were used for preliminary fragmentation schemes construction. The lowest substrate background activity has been established for monolithic silica gel thin layer and aluminium backplate modified Si 60 thin layer. In the case of Raman spectroscopy pre-treated thin layer or conventional chromatographic thin layer Si 60, the both, high background signal intensity and impossibility to construct negative ions surface image, were observed. Diterpenoic acids studied serve the similar mass spectrum but ToF-SIMS coupled with liquid chromatographic separation brings new impact to the positive identification of analytes studied.

The development of endotension is associated with increased transmission of pressure and serous components in porous expanded polytetrafluoroethylene stent-grafts: Characterization using a canine model

This study used a canine model of abdominal aortic aneurysms (AAAs) to compare intra-aneurysmal pressure and thrombus formation after exclusion with Dacron and expanded polytetrafluoroethylene (ePTFE) stent-grafts. Prosthetic AAAs with implanted strain-gauge pressure transducers were treated by stent-graft exclusion using Food and Drug Administration-approved devices in 10 mongrel dogs: five Dacron (AneuRx) and five ePTFE (original Excluder). Intra-aneurysmal pressure was measured over 4 weeks after AAA exclusion and indexed to the systemic pressure, represented as a percentage of a simultaneously obtained systemic pressure (value = 1.0). Magnetic resonance imaging (MRI) of the intra-aneurysmal thrombus was performed at 1, 2, and 4 weeks after exclusion and expressed as a signal-to-noise ratio (S:N) to control for background signal intensity. Comparisons of pressures and S:N between the two stent-grafts was analyzed with the Student's t test. Intra-aneurysmal thrombus was characterized histologically. In animals excluded with both Dacron and ePTFE stent-grafts, the intra-aneurysmal pressure was nonpulsatile and reduced to <30% of systemic pressure. Significantly greater pressure transmission was observed after AAA exclusion using ePTFE compared with Dacron stent grafts (systolic pressure: ePTFE, 0.28 +/- 0.12 vs Dacron, 0.11 +/- 0.02, P < .001; mean pressure: ePTFE, 0.16 +/- 0.08 vs Dacron, 0.06 +/- 0.02, P < .001). MRI confirmed the absence of perfusion in all aneurysms. The T1-weighted signal intensity remained persistently elevated (S:N at 1 week, 2.7 +/- 0.4 vs 2 weeks, 4.0 +/- 0.2 vs 4 weeks, 5.4 +/- 1.3) in ePTFE-treated intra-aneurysmal thrombus, suggesting an absence of thrombus organization. In contrast, progressive evolution of T1 signal intensity in aneurysms excluded by Dacron stent-grafts was consistent with maturation from intact red blood cells (S:N at 1 week, 3.3 +/- 0.4) to methemoglobin (S:N at 2 weeks, 6.1 +/- 0.8), and then hemosiderin and ferritin (S:N at 4 weeks, 2.4 +/- 0.5). Histologically, ePTFE-excluded aneurysms contained poorly organized thrombus with red blood cell fragments and haphazardly arranged fibrin deposition indicative of active remodeling and continued influx of transudated serum. In aneurysms excluded by Dacron stent-grafts, dense, mature collagenous connective tissue and organized fibrin were present, indicative of greater thrombus organization. Stent-graft treatment reduces intra-aneurysmal pressure to <30% of systemic pressure when no endoleak is present; however, significantly greater pressure is present in aneurysms treated with porous ePTFE stent-grafts than Dacron grafts. Histologic and MRI imaging analysis suggest that active transudation of serous blood components may be contributing to this increased intra-aneurysmal pressure.